3.2. Synthetic Procedures and Characterisation of Compounds

Synthesis of methyl (5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoyl)-L-prolinate (1b):

Compound 1a (2 gm, 6.12 mmol, 1 equiv.), obtained according to a literature procedure [12], was dissolved in dry DCM and transferred to a round bottom flask. Oxalyl chloride (2.33 gm, 18.36 mmol, 3 equiv.) was added to the solution, followed by a catalytic amount of dry DMF (2–3 drops) and left under a magnetic stirrer, while the development of gas was observed. After one hour, dry toluene (15 mL) was added to the solution that was evaporated under a vacuum using a rotary evaporator. The obtained orange residue was dissolved in dry DCM and added dropwise at 0 °C to a solution made of triethylamine (1.86 gm, 18.36 mmol, 3 equiv.) and commercially available (S)-proline methyl ester (1.4 gm, 9.18 mmol, 1.5 equiv.) in dry DCM. The reaction mixture was left under a magnetic stirrer overnight at room temperature under an N2 atmosphere until TLC showed completion of the reaction. The reaction mixture was diluted by the addition of DCM (20 mL) and subsequently washed with 1 N HCl (2 × 70 mL) and brine (2 × 70 mL). The organic phase was dried over MgSO4 and evaporated under a vacuum using a rotary evaporator to give a yellow oil. The crude of the reaction was subsequently purified by column chromatography on silica gel (mobile phase: from EtOAc, 100, to EtOAc/MeOH, 98/2, v/v) to give intermediate 1b (0.900 g, reaction yield: 36%), obtained as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: as a mix of rotamers, 7.69/7.65 (s, 1H), 6.87/6.79 (s, 1H), 4.75–4.69 (m, 1H), 4.17–4.14 (t, J = 6.4, 2H) 4.13–4.00 (m, 1H), 3.97/3.93 (s, 3H), 3.80/3.66 (s, 3H), 3.70/3.53 (s, 3H), 3.33–3.29 (m, 1H), 3.21–3.17 (m, 1H), 2.58–2.52 (m, 3H), 2.38–2.30 (m, 1H), 2.24–2.18 (m, 3H), 2.12–1.89 (m, 4H). 13C NMR (101 MHz, CDCl3) δ: as a mix of rotamers, 173.2/173.3, 172.6/172.4, 154.8/154.2, 148.5/148.3, 137.3/137.1, 127.4/127.2, 110.2/109.5, 108.2/108.1, 68.1, 60.6, 58.5, 56.7/56.5, 52.4/52.3, 51.5, 48.3, 46.2, 31.0, 30.3/29.6, 24.5.

Synthesis of methyl (S)-4-((7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanoate (1c):

Intermediate 1b (900 mg, 2.12 mmol, 1 equiv.) was dissolved in EtOH in a hydrogenator vial, and a catalytic amount of Raney-Nickel, slurry in H2O (100 mg), was added to the solution. The vial was placed in position in a Parr hydrogenation system and reacted under the pressure of H2 (40 psi) until TLC showed total consumption of the starting material. The reaction mixture was subsequently filtered on a Celite path, washing with DCM. The collected organic solvent was evaporated under a vacuum using a rotary evaporator to give pure 1c. If the reaction resulted in a mixture of unclosed and closed products, the crude of the reaction was refluxed in toluene after filtration of the catalyst to give conversion to the desired product 1c (0.800 g, reaction yield: 95%), obtained as an orange oil. 1H NMR (400 MHz, CDCl3) δ: 8.91 (s, 1H), 7.42 (s, 1H), 6.52 (s, 1H), 4.04 (td, J = 3.08, 5.92 Hz, 3H), 3.87 (s, 3H), 3.71–3.78 (m, 1H), 3.67 (s, 3H), 3.55–3.62 (m, 1H), 2.70–2.76 (m, 1H), 2.53 (t, J = 7.05 Hz, 2H), 2.15–2.09 (m, 2H), 1.96–2.02 (m, 3H). 13C NMR (101 MHz, CDCl3) δ: 173.5, 171.2, 165.3, 151.5, 146.6, 129.8, 119.3, 112.3, 105.1, 67.8, 56.9, 56.2, 51.7, 47.3, 30.2, 26.5, 24.1, 23.6.

Synthesis of (S)-4-((7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanoic acid (1d):

Intermediate 1c (790 mg, 2.18 mmol, 1 equiv.) was dissolved in methanol (60 mL), and aqueous 1M NaOH solution (excess) was added to the reaction mixture that was stirred overnight at room temperature until TLC showed total consumption of the starting material. A rotary evaporator was used to evaporate the solvent. The obtained residue was dissolved in H2O (30 mL), and citric acid 1M aqueous solution was added to the solution until pH 3 was reached. The aqueous phase was extracted with EtOAc (2 × 50 mL) and the combined organic phases were evaporated under a vacuum using a rotary evaporator, affording pure 1d (0.700 g, reaction yield: 92%), obtained as a white solid. 1H NMR (400 MHz, MeOH-d4) δ: 7.27 (s, 1H), 6.57 (s, 1H), 4.10–4.05 (m, 1H), 3.99 (t, J = 6.4Hz, 2H), 3.75 (s, 3H), 3.69–3.59 (m, 1H), 3.53–3.42 (m, 1H), 3.24–3.17 (m, 2H), 2.59–2.51 (m, 1H), 2.42 (t, J = 7.2Hz, 2H), 2.03–1.90 (m, 4H). 13C NMR (101 MHz, MeOH-d4): 176.8, 172.4, 167.6, 153.5, 147.9, 132.3, 120.0, 113.1, 106.5, 69.1, 58.4, 56.6, 31.2, 27.0, 25.5, 24.6.

Synthesis of dilactam derivatives 7a and 8a:

The Boc-protected intermediates 2a and 2b, prepared accordingly to a reported literature procedure [7] (1.2 equiv.), were dissolved in MeOH (7 mL), and HCl 4M in dioxane (7 mL) was added to the solution that was left under a magnetic stirrer for 2 h until TLC showed completion of the reaction. The reaction mixture was then evaporated under a vacuum using a rotary evaporator, giving the corresponding deprotected intermediates.

Compound 1d (52 mg, 1 equiv.) was dissolved in DMF (7 mL), and EDCI (59 mg, 2.4 equiv.) and DMAP (50 mg, 3 equiv.) were added to the solution that was left to stir in N2 atmosphere for 30 min. Either the deprotected compound 2a (60 mg, 0.123 mmol, 1.1 equiv.) or 2b (55 mg, 0.124 mmol, 1.1 equiv.) was added to the reaction mixture and left to stir overnight at room temperature in the N2 atmosphere. The reaction did not complete and was quenched by adding H2O (10 mL). The aqueous phase was then extracted with EtOAc (3 × 10 mL). The organic phase was sequentially washed with citric acid 0.1 M aqueous solution (10 mL), saturated NaHCO3 aqueous solution (10 mL), and brine (10 mL). The collected organic phase was dried over MgSO4 and evaporated under a vacuum using a rotary evaporator. The crude of the reaction was purified by column chromatography on silica gel (mobile phase: DCM/Acetone, 50/50, v/v) to give the final pure products.

(S)-4-(4-((7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanamido)-1-methyl-N-(2-(thiomorpholine-4-carbonyl)benzofuran-5-yl)-1H-pyrrole-2-carboxamide (7a)

Obtained 0.040 g (reaction yield: 43%) as a yellow oil. 1H NMR (400 MHz, Acetone-d6) δ 9.25 (s, 1H), 9.22 (s, 1H), 9.12 (s, 1H), 8.21 (d, J = 2.01 Hz, 1H), 7.67 (dd, J = 8.94 Hz, 2.14 Hz, 1H), 7.48 (d, J = 9.06 Hz, 1H), 7.31 (s, 1H), 7.27 (d, J = 1.01 Hz, 1H), 7.19 (d, J = 1.76 Hz, 1H), 6.91 (d, J = 2.01 Hz, 1H), 6.74 (s, 1H), 3.93–4.11 (m, 6H), 3.89 (s, 3H), 3.80 (s, 3H), 3.54–3.65 (m, 1H), 3.43–3.52 (m, 1H), 2.68–2.75 (m, 5H), 2.47 (t, J = 7.18 Hz, 2H), 1.99–2.04 (m, 6H). 13C NMR (101 MHz, MeOH-d4) δ 173.7, 172.3, 167.6, 162.4, 161.8, 153.5, 153.0, 150.2, 147.8, 136.0, 132.3, 128.4, 124.5, 123.3, 122.6, 121.2, 119.8, 115.6, 113.1, 113.0, 112.7, 106.5, 106.4, 74.1, 69.2, 58.4, 56.5, 44.0, 36.9, 33.5, 30.7, 29.5, 27.0, 26.3, 24.6. HRMS (ESI, m/z): calc. for C36H39N6O8S1 ([M] + H) + 715.2545 found 715.2554.

(S)-N-(2-(dimethylcarbamoyl)benzo[b]thiophen-5-yl)-4-(4-((7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide (8a)

Obtained 0.040 g (reaction yield: 46%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.24 (d, J = 1.76 Hz, 1H), 7.66 (d, J = 8.81 Hz, 1H), 7.46 (dd, J = 8.69 Hz, 1.89 Hz, 1H), 7.37 (s, 2H), 7.04 (s, 1H), 6.81 (s, 1H), 6.48 (s, 1H), 3.95–4.01 (m, 1H), 3.85–3.92 (m, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.66–3.74 (m, 1H), 3.50–3.59 (m, 1H), 3.17 (br. s., 6H), 2.68 (dd, J = 6.04 Hz, 4.28 Hz, 1H), 2.29–2.37 (m, 2H), 1.83–2.12 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 170.6, 170.1, 165.5, 164.9, 160.2, 151.4, 146.2, 139.2, 138.1, 135.8, 135.4, 130.2, 125.7, 123.3, 122.4, 121.6, 119.8, 119.25, 115.7, 112.4, 106.7, 104.6, 68.0, 57.0, 56.1, 47.2, 36.8, 31.8, 32.6, 29.3, 26.2, 23.6. HRMS (ESI, m/z): calc. for C34H37N6O7S1 ([M] + H) + 673.2439 found 673.2447.